Epidermal Growth Factor (EGF)
Background Human epidermal growth factor (EGF), a 6.2kDa protein containing 53 amino acid residues, is the ligand for the EGF-receptor (EGFR). This ligand-receptor interaction can induce a host of mitogenic changes within the cell ranging from proliferation and differentiation to cell survival and protection from environmental hazards. EGF was discovered by Stanley Cohen and Rita Levi-Montalcini in 1996, a discovery that awarded them the nobel prize in Physiology and Medicine (1) . EGF Family, Mechanisms and Therapies The EGF ligand is the original member of the EGF family, although, other members have similar structural and functional characteristics (1). Among family members, there are 6 highly conserved cysteine residues that can form disufide bonds. Also conserved are a single glycine and arginine. The disulfide bridges help contort the EGF ligand (or any other family member) in such a way that it creates a high affinity binding interaction with the cell surface EGFR (1). Members of the EGF family are: heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha (TGF-alpha), amphiregulin (AR), Epiregulin (EPR), Epigen, betacellulin (BTC) and neuregulins 1-4 (NRGx )(1). EGF or other family member ligand, has a high affinity for its cell surface receptor EGFR. Once ligand is bound to the receptor, a cascade of signaling events occurs via the intrinsic protein-tyrosine kinase activity of the receptor (1) (2). Events that can be caused by EGFR activation include; rises in intracellular calcium levels, increased glycolysis and protein synthesis, increased cellular proliferation and motility. Because of its role in cell migration and survival, the EGFR pathway has recently been exploited as a hot topic in cancer research (3) . Some cancer patients, specifically lung cancer patients, harbor mutations within the EGFR gene region. Recent research has attempted to target these mutations as means of chemotherapeutics (3). The anti-cancer drug, Erlotinib, specifically targets the T790M mutation within the receptor, blocking it and disallowing the further activation of the receptor which can ultimately aid in the killing of cancer cells (4) . Recombinant EGF Human recombinant EGF, isolated from E.coli, is used to study many different pathways in cancer and metastasizing diseases. E.coli are generally considered optimal organisms for the amplification of certain recombinant proteins because of their hardiness and ability to exponentially grow to produce mass amounts of targeted protein. Some proteins, such as membrane bound proteins (like EGFR), would not be particularly suitable for growth in E.coli. The extracted human EGF gene is placed in a vector or plasmid of choice, usually paired with an antibiotic resistance gene and inducible promoter sequences, is then transfected into host E.coli cells. The E.coli colonies are selected via antibiotics and allowed to proliferate until optimal density is acquired. Once 'grown up', protein lysate mixtures are extracted from the E.coli cells. This protein mixture is then placed in an affinity column that has anti-GFP bound antibodies. The human EGF is then eluted off the column and retained. Further purification is required to ensure quality. Millipore, Sigma Aldrich and Lifesciences (Invitrogen) report over a 98% purity based on SDS-PAGE analysis (5) (6) (7) . Recombinant EGF can also be used to study these signaling pathways in a variety of tissues and cell types. Related tissues effected by EGF are; brain, neurons, skeletal muscle, prostate, liver, pancreas, lung, tongue, skin, kidney and the trachea (5). The cell types that can be studied via the use of EGF at a mitogen are; fibroblasts, glial cells, mammary epithelial cells, vascular and corneal endothelial cells, bovine granulosa, rabbit chondrocytes, HeLa cells and SV40-3T3 cells (5). Research has also shown the use of recombinant EGF as a topical skin treatment of chronic wounds (venous ulcers) in which treatment reduced ulcer size in 73% of patients vs 33% given a placebo (8). References #http://en.wikipedia.org/wiki/Epidermal_growth_factor #Carpenter G, King L, Jr., & Cohen S (1979) Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J Biol Chem 254(11):4884-4891 #da Cunha Santos G, Shepherd FA, & Tsao MS (2011) EGFR mutations and lung cancer. Annu Rev Pathol 6:49-69 #Sos ML, et al. (2009) PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res 69(8):3256-3261 #http://www.sigmaaldrich.com/catalog/product/sigma/e9644?lang=en&region=US #http://www.millipore.com/catalogue/item/gf144 #http://www.lifetechnologies.com/order/catalog/product/PHG0311 #Falanga V, et al. (1992) Topical use of human recombinant epidermal growth factor (h-EGF) in venous ulcers. J Dermatol Surg Oncol 18(7):604-606